Each day a cell accumulates nearly 20,000 DNA lesions, which, if allowed to persist, can lead to genomic instability and cancer. Single-base DNA lesions are primarily removed and repaired by the base excision repair (BER) pathway. DNA polymerase beta (Pol ß) performs the gap-filling reaction of BER. Pol ß is mutated in 30 % of human tumors, suggesting a role for Pol ß in tumorigenesis. Pol ß Y265C is a human gastric-tumor associated variant that has been shown to have slow DNA synthesis activity /in vitro/. In a mouse cell line, expression of Pol ß Y265C resulted in increased spontaneous mutagenesis. To test the hypothesis that Pol ß Y265C can drive tumorigenesis, we developed a knock-in mouse model for Pol ß Y265C in the 129SV/J-C57BL6 mouse hybrid background. 60 % of the homozygous mutant mice died within a few hours of birth. We introduced the Pol ß Y265C mutation in a p53-deficient background and found that the Y265C homozygous mutant mice are born and survive. Thus, the deletion of p53 rescues post-natal lethality of the Y265C homozygous mutant mice. We observed that, in the p53 deficient background, as compared to the wild-type (WT) mice (165 days), the mean survival of the heterozygous and homozygous Y265C mutant mice was reduced (115 days and 116 days respectively). The WT mice predominantly died from lymphoma. The heterozygous (C/+) mice not only had a decreased latency for lymphoma development but also developed gastrointestinal (GI) adenocarcinomas (7 %). Interestingly, 25 % of the homozygous mutant (C/C) mice developed neuroblastoma. These data suggest that Pol ß Y265C is capable
of driving tumorigenesis in the GI tract and the brain. To understand the mechanism by which Pol ß Y265C induces tumorigenesis, we harvested fibroblasts (MEFs) from 12.5 day-old embryos. We found that the heterozygous and homozygous mutant MEFs proliferate at a significantly slower rate than the WT MEFs at 20 % oxygen. However, at 5 % oxygen, no difference in proliferation rates was observed between the three MEF cell lines. By immunofluorescence microscopy, we found that the homozygous mutant cells accumulated more gamma-H2AX, foci. gamma- H2AX is a surrogate marker for DNA double-stand breaks. Hence, the immunofluorescence data suggest that the
expression of Pol ß Y265C results in the accumulation of DNA double-strand breaks. Analysis of metaphase spreads by Giemsa staining showed that the homozygous mutant cells accumulated more chromatid breaks as well as chromosome fragments and fusions than the WT cells. Taken together, the data from the MEF study demonstrate that the expression Pol ß Y265C leads to the accumulation of DNA double strand breaks and chromosomal aberrations. In the presence of p53, cells harboring Pol ß Y265C induced genomic instability undergo cell death. However, our data show that in the p53-deficient background, the cells can survive and proliferate at low oxygen and thereby
drive tumorigenesis. These findings are significant because they are the first to use a mouse model to provide mechanistic insight into the role of DNA polymerase ß in the onset of cancer.